Project Summary: Macrophage Determinants of Retinal Regeneration The objective of this proposal is to understand microglia and macrophage ontogeny, dynamics, and endogenous roles in retinal degeneration and regeneration using the zebrafish as a model, a vertebrate organism capable of robust retinal regeneration following a variety of insults. Humans do not possess this regenerative capacity both in contexts of acute retinal damage or in neurodegenerative diseases of the retina, although microglia and macrophages are active participants in the response to neuronal death, and macrophages are able to drive wound healing in peripheral tissues. Further, in some contexts, microglia and macrophages appear to contribute to pathology, though modulation of such pathological contributions has not been achieved. In order to provide future therapeutic and regenerative strategies to support endogenous microglia/macrophage-specific mechanisms that positively regulate the outcome of regeneration and/or impart the capacity to perform such functions through strategic manipulation, more foundational knowledge is required. We propose that a thorough understanding of microglia and macrophage functions in a system of robust retinal regeneration (specifically, the zebrafish) will reveal microglia and macrophage functions that can ultimately be harnessed to mitigate neuroinflammation and support attempts at retinal regeneration in humans. Our published and preliminary data indicate that in zebrafish, both retinal resident microglia and extra- retinally derived macrophages are present in degenerating and regenerating retinal tissue following an acute cytotoxic lesion, and these microglia/macrophages intimately interact with regenerative Mller glia, the cell type acting as the source of regenerated neurons. Transcriptional profiling of microglia/macrophages during active Mller glia-mediated regeneration indicates functional changes in microglia/macrophages compared to steady- state. Our preliminary data also indicate that altering microglial phenotype to a pro-inflammatory state may contribute to neuronal degeneration. We hypothesize that microglia and macrophages perform crucial functions in shaping the outcome of retinal degeneration and regeneration. The following Specific Aims will test this hypothesis. 1. Determine the extent and duration of microglia and macrophage heterogeneity following retinal injury. 2. Determine how pro-inflammatory macrophages affect retinal degeneration and regeneration. 3. Determine endogenous function(s) of microglia and macrophages in retinal regeneration. Findings from this proposal will (I) provide crucial knowledge and tools towards future research to identify microglia vs. macrophage-specific molecular mechanisms underlying retinal degeneration and successful regeneration and (II) facilitate comparative studies to identify key factors and mechanisms that determine outcome (pathology vs. regeneration) following retinal damage in zebrafish compared to mammals. Collectively, this new knowledge will provide foundations towards successful design and application of therapeutic strategies for human retinal damage and disease.